Transcriptomic evidence of lung repair in paediatric ARDS survival

Dear Editor, Acute respiratory distress syndrome (ARDS) is triggered mostly by viral or bacterial infections. As the lung and immune systems are still developing in childhood, the pathophysiology of paediatric ARDS (PARDS) may differ substantially from ARDS in adults, leading to substantial differences in clinical outcomes.1–3 The pathological process of ARDS is divided into acute exudative, proliferative and fibrotic phases,3 which can be superimposed,with profibrotic pathways being triggered as early as the first day of the illness.4,5 The efficacy of veno-venous (VV) extracorporeal membrane oxygenation (ECMO) in patients with ARDS remains controversial6,7 because the mortality of ARDS remains high and is closely associated with abnormal lung repair and severe complications of ECMO.8 Current pathological findings are based on autopsies or biopsies of non-surviving patients with severe ARDS. Comprehensive evidence of reparativemechanisms in surviving ARDS is currently lacking. Here we present a case report of a 10-year-old girl who survived ARDS with an air leak under ECMO support. We performed a single-cell RNA sequencing (scRNA-seq) analysis of the lung biopsy sample taken from the surgical repair of the pneumothorax and tracked dynamic transcriptomic changes in peripheral blood immune cells. These preliminary data provided insight into the immune microenvironment that favours lung repair in PARDS. A previously healthy 10-year-old girl presented with fever, left chest pain and lethargy (D1; Figure 1A). Chest computed tomography (CT) showed diffuse groundglass opacity (GGO) in the right upper and lower lobes (Figure 1B). As dyspnoea worsened, the patient was intubated on D4. Methylprednisolone was administered systemically from D11, and the patient’s body temperature gradually returned to normal (Supplementary Figure 1A). A repeat chest CT suggested diffuse progression of the GGO (Figure 1C). Neither respiratory pathogens (Supplementary Table S1) nor streptolysin O was detected. On D14, her respiratory distress worsened, and chest

radiography showed severe subcutaneous emphysema and pneumomediastinum. VV ECMO was initiated on D23, and right-sided closed thoracic drainage was performed.
Post-ECMO, a CT scan showed bilateral pneumothorax, diffuse subcutaneous emphysema, pneumomediastinum and bilateral atelectasis ( Figure 1D). Moreover, nextgeneration sequencing showed that the diversity and abundance of residential bacteria were sharply decreased (Supplementary Figure 1B). With no improvement in the pneumothorax ( Figure 1K), we performed video-assisted thoracoscopic surgery with ECMO support. The right lung was tough, with some ulcers on the visceral pleura and sticky purulent exudate between the lobes ( Figure 1H,I). Each lobe was unevenly re-inflated after high-pressuring single-lung ventilation ( Figure 1J). Two ruptured emphysematous bullae were identified on the edge of the upper lobe. A small portion of tissue from the lesion was collected for biopsy before the bullae were closed by double ligation. Haematoxylin and eosin staining of the tissue showed characteristics of the proliferative phase of ARDS ( Figure 1 M-S). The right lung re-inflated after surgery ( Figure 1L). The patient was successfully weaned off ECMO on postoperative day 6 (D37). A chest CT on D46 showed no signs of subcutaneous emphysema or pneumomediastinum ( Figure 1F). A chest CT at 6 months of follow-up showed that the lung lesions had almost completely disappeared ( Figure 1G).
scRNA-seq analysis of lung tissues suggested that genes of major histocompatibility complex (MHC) class II, MHCI and inflammation were decreased in the macrophage subsets of the ARDS subject versus controls, while genes related to lung-repair, lipid-metabolism and phagocytosis were upregulated (Supplementary Figure 2; Figure 2A-D). In the ARDS subject, extracellular matrix production and metabolism-related genes, including CCL18, ELN and DCN, were downregulated in Early B-Cell Factor 1 (EBF1) relatively highly expressed fibroblasts (EBF1 + fibroblasts) and lipofibroblasts. Genes associated with lung repair, including EGR3, TGFB1, TGFBI, AREG, FGF7, IL33, VEGFA, PDGFRA, VCAN, CTGF and TIMP1, were upregulated in the fibroblast subsets ( Figure 2D; Supplementary Table S2). Furthermore, enhanced cell-cell interactions among ficolin 1 (FCN1) relatively highly expressed macrophages (FCN1 + macrophages), secreted phosphoprotein 1 (SPP1) relatively highly expressed macrophages (SPP1 + macrophages), lipofibroblasts and mesothelial cells were observed in the ARDS subject ( Figure 2E). Therefore, multiple cells exhibiting a lung repair phenotype may have contributed to the survival of this child with ARDS.
Next, we assayed the dynamic transcriptome of myeloid cells during ECMO (Supplementary Figure 3; Figure 3A,B). The mitochondrial and MHCII-related genes showed an upward trend in the ARDS subject from ECMO initiation to intensive care unit (ICU) discharge ( Figure 3C-F). Interferon-stimulated gene (ISG) expression remained at low levels before ECMO weaning ( Figure 3F). Interferon-and inflammation-related genes were downregulated at ECMO weaning versus initiation ( Figure 3F).
A set of transcripts was screened in mature neutrophils that were positively or negatively associated with disease improvement ( Figure 4A). The upregulated transcripts in this set were enriched in the biological process of mitochondrial functional improvement ( Figure 4B,C). We found that apoptosis rates were significantly decreased after disease improvement, suggesting that the high expression of mitochondrial functional genes is not caused by excessive cellular apoptosis (Supplementary Figure 4). In contrast, the expression of genes necessary for the migration and adhesion, including FLNA, CD44, MYADM and AQP9, were downregulated ( Figure 4C). Meanwhile, HCK, FGR and PECAM1, which are related to the phagocytosis, gradually returned to normal levels after ECMO weaning ( Figure 4C). The expression of ISG and MHCIrelated transcripts remained lower during ECMO but was upregulated at ICU checkout ( Figure 4D).
We also found megakaryocytes communicated with haematopoietic stem cells via transforming growth factorbeta signalling to reduce ECMO-associated bleeding risk (Supplementary Figure 5).
As an invasive examination that rarely alters clinical treatment strategies, lung biopsy is not widely utilized in ARDS patients, which also makes it difficult to explore the cellular functionality and communication characteristics during the pulmonary injury repair phase. To the best of our knowledge, this is the first study to describe dynamic changes in peripheral blood cells and lung tissue at the single-cell transcriptomic level in patients with PARDS with ECMO support.
This study had several limitations. First, substantial immunological maturation occurs between ages 1 and 10 years, which may limit the generalizability of our study findings to other age groups. Second, we cannot fully differentiate between the ARDS and ECMO effects on neutrophils and other immune cells. Third, we acknowledge that the ideal samples should be collected from individuals of the same sex and age group. However, due to the scarcity of samples, we can only use the best samples available from children as the control group. Although this was a quick glance, these data provided insight into the characteristics of the immunological microenvironment and lung tissue repair required for the recovery of children with ARDS.  Gene ontology (GO) analysis of screened genes in C (chi-squared test, p < 0.05). The colour of the dots from blue to red indicates the low to high expression rate of genes that were included in the corresponding GO term in each sample. Dot size is relative to the score of the GO term in each sample. (E) Violin plots of selected genes that were involved in mitochondrial function and major histocompatibility complex class II from ECMO initiation, ECMO weaning off, ICU checkout and healthy controls. (F) Violin plots of selected genes that were involved in interferon-stimulated genes, interferon receptors, MHCI molecules, pro-inflammatory and anti-inflammatory factors from ECMO initiation, ECMO weaning off and ICU checkout.

F I G U R E 4
Dynamic transcriptomic characteristics of peripheral mature neutrophils. (A) Heatmap of genes screened out from mature neutrophils of extracorporeal membrane oxygenation (ECMO) initiation, ECMO weaning off, ICU checkout and healthy control. (B) Gene ontology (GO) analysis of screened genes in A (chi-square test, p-value < 0.05). The colour of the dot from blue to red indicates the low to high expression rate of genes that were included in the corresponding GO term in each sample. Dot size is relative to the score of the GO term in each sample. (C) Violin plots of selected genes that were involved in mitochondrial function, migration, adhesion, phagocytosis, metallopeptidase inhibitor and regulation of endothelial cell differentiation from ECMO initiation, ECMO weaning off, ICU checkout and healthy control. (D) Violin plots of selected genes that were involved in interferon-stimulated genes, interferon receptors, oxidative stress, anti-bacterial function, anti-inflammatory, MHCI molecules and degranulation from ECMO initiation, ECMO weaning off and ICU checkout.

A C K N O W L E D G E M E N T S
We thank all patients who participated in the trial and their parents or guardians. This study was funded by the National Natural Science Foundation of China (nos. 82172109, 82070001), Key Project of the 8th Center of Chinese PLA General Hospital (no. 2021ZD008), National Key R&D Program of China (no. 2021YFC2701700) and Natural Science Foundation of Tianjin, China (nos. 21JCZDJC00430, 21JCQNJC00510). Some of the images were created using BioRender.com.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
No disclosures were reported.

D ATA AVA I L A B I L I T Y S TAT E M E N T
Multi-omics data are openly available in GEO bank (GSE223793) at https://www.ncbi.nlm.nih.gov/geo/. Clinical data are available from the corresponding authors upon reasonable request and with the permission of the institution.